Initiation of inflammation begins with an inflammatory response and leads to the activation of neutrophils, granulocytes, monocytes, macrophages, as well as other immunomodulatory cells. This may result in a topical or systemic inflammatory cascade involving inflammatory cytokines and mediators, such as interleukins (ILs), Tumor necrosis factor alpha (TNFα), and prostaglandins. This complex inflammatory mediated cascade triggers a whole range of responses, such as cellular chemotaxis and endothelial injury and leads to the recruitment of additional cells from the innate and adaptive immune systems.
The skin serves as an important boundary between the internal body and the environment, preventing contact with potentially harmful pathogens. In the case of antigen/pathogen penetration, an inflammatory response is often induced to eliminate the antigen. This response leads to a dermal infiltrate that consists predominantly of T cells, polymorphonuclear cells, and macrophages.
The inflammatory response is not necessarily associated with external stimuli, or may be caused by a non-harmful environmental substances (in case of allergies). In both cases an over-expression of proinflammatory cytokines without proper controls leads to inflammation which is the hallmark of topical and systemic inflammation generally, as well as a variety of inflammatory diseases and disorders. Inflammation is associated with a variety of disorders such as eczema and dermatitis, including for example, atopic dermatitis, seborrheic dermatitis, dyshidrotic eczema, nummular dermatitis, stasis dermatitis, allergic dermatitis, psoriasis, pruritis, multiple sclerosis, cutaneous inflammation, cicatricial pemphigoid, scleroderma, hidradenitis suppurativa, toxic epidermal necrolysis, acne, osteitis, graft vs. host disease (GvHD), pyroderma gangrenosum, and Behcet's Syndrome.
Over production of proinflammatory cytokines has been implicated in many inflammatory and autoimmune diseases. For example, the secretion of cytokines such as TNFα and Interleukin-23 (IL-23), which stimulates survival and proliferation of Th17 cells, are highly associated with psoriasis, where IL-6 is required for Th17 development in addition to its general role as proinflammatory cytokine. Other cytokines like IL-12 and IP-10 are initiators and involves in Th1 pathway which is typical to psoriasis and other autoimmune diseases. IL-5, a cytokine that increases the production of eosinophils, is over-expressed in asthma resulting in accumulation of eosinophils in the asthmatic bronchial mucosa, a hallmark of allergic inflammation. IL-4 and IL-13 are known mediators of the hypercontractility of smooth muscle found in inflammatory bowel disease and asthma. Additionally, as discussed further below, inflammatory cytokines have been shown to be implicated in, by way of example, psoriasis, multiple sclerosis, arthritis, ischemia, septic shock, and organ transplant rejection.
Similarly, granulocyte macrophage-colony stimulating factor (GM-CSF) is a regulator of maturation of granulocyte and macrophage lineage population and has been implicated as a key factor in many inflammatory and autoimmune diseases. For example, antibodies that inhibit GM-CSF secretion have been shown to ameliorate autoimmune disease.
Thus, development of therapeutics that reduce secretion of proinflammatory cytokines and/or regulate immunomodulators would be beneficial in alleviating topical and systemic inflammation generally, as well as a host of inflammatory and/or autoimmune diseases as discussed herein. Several lines of evidence point to modulators of PKC isoforms as useful in achieving these results.
Several in vivo studies have shown the involvement of T helper (Th) 17 cells as well as secretion of cytokines such as interleukins and TNFα, by skin associated cells such as keratinocytes, dendritic and T helper cells, as key players in the development of the inflammatory response involved in the pathogenesis of psoriasis and other autoimmune inflammatory diseases. The secretion of cytokines such as TNFα and Interleukin (IL)-23, which stimulates survival and proliferation of Th17 cells, also serves as a key master cytokine regulator for these diseases. (Fitch et al. (2007) Curr Rheumatol Rep. 9:461-7). Th17 cells within dermis in turn, induce secretion of IL-17A and IL-22. IL-22, in particular, derive keratinocyte hyperproliferation and augment the inflammatory response in psoriasis (Fitch et al. (2007) Curr Rheumatol Rep 9:461-7).
The protein kinase C (PKC) family represents a group of phospholipid dependent enzymes catalyzing the covalent transfer of phosphate from ATP to serine and threonine residues of proteins. The family is currently considered as composed of at least 12 individual isoforms which belong to 3 distinct categories based on their activation by calcium ion(s) and other factors. The PKC family consists of at least ten members, usually divided into three subgroups: classical, novel and atypical PKCs (FIG. 1). The specific cofactor requirements, tissue distribution, and cellular compartmentalization suggest differential functions and the tuning of specific signaling cascades for each isoform. Thus, specific stimuli can lead to differential responses via isoform specific PKC signaling regulated by their factors, such as: expression, localization, and/or phosphorylation status in particular biological settings. PKC isoforms are activated by a variety of extracellular signals and, in turn, modify the activities of cellular proteins including receptors, enzymes, cytoskeletal proteins, and transcription factors. Accordingly, the PKC family plays a central role in cellular signal processing including regulation of cell proliferation, differentiation, survival and death.
PKCα, which is highly abundant in skin, is the major conventional, Ca2+ responsive, PKC isoform in epidermis and it was initially the only cPKC detected in the keratinocytes in vitro and in vivo (Dlugosz et al. (1992) Biomed Pharmacother 46:304; Wang et al. (1993) J Cancer Res Clin Oncol 119:279-287). Therefore, PKCα had been proposed as a key player in Ca2+ induced differentiation (Denning et al. (1995) Cell Growth Differ 6:149-157; Dlugosz et al. (1992) Biomed Pharmacother 46:304). Being in epidermis and mainly restricted to suprabasal layers (Denning et al. (2004) Int J Biochem Cell Biol 36:1141-1146), PKCα is involved in cell cycle withdrawal and primarily associated with the keratin cytoskeleton and desmosomal cell-cell junctions (Jansen et al. (2001) Int J Cancer 93:635-643; Tibudan et al. (2002) J Invest Dermatol. 119:1282-1289). Since, upon exposure to the classical PKC activator TPA (12-O-tetradecanoylphorbol-13-acetate), spinous markers were suppressed, PKCα was thought to be largely responsible for the shift from spinous to granular differentiation as a result of TPA activation (Dlugosz and Yuspa (1993) J Cell Biol 120:217-225; Lee et al. (1998) J Invest Dermatol 111:762-766; Matsui et al. (1992) J Invest Dermatol 99:565-571; Punnonen et al. (1993) J Invest Dermatol 101:719-726). Indeed, blocking PKCα activity or its synthesis by antisense oligonucleotides appeared to abolished granular markers and revive spinous markers like K1 and K10. Likewise, implementation of dominant negative PKCα appeared to restore the (late) spinous marker involucrin (Deucher et al. (2002) J Biol Chem 277:17032-17040). Accordingly, defective differentiation in skin cancer (Tennenbaum et al. (1993) Cancer Res 3:4803-4810; Tomakidi et al. (2003) J Pathol 200:298-307) correlates with elevated PKCα activity, also observed in tumor cells in vitro (Dlugosz et al. (1992) Biomed Pharmacother 46:304; Yang et al. (2003) J Cell Physiol. 195:249-259). However, over-expression of PKCα in normal human keratinocytes did not appear to alter their differentiation pattern (Deucher et al. (2002) J Biol Chem 277:17032-17040). The influence of PKCα on the cellular traffic and membrane recruitment of β1-integrin during migration (Ng et al. (1999) EMBO J. 18:3909-3923) may well promote both wound reepithelialization and tumor cell invasion.
Over-expression of PKCα in transgenic mice has appeared to induce a striking inflammatory response, increased epidermal thickening and edema correlated with neutrophil infiltration, multiple micro-abscesses, and a marked increase of inflammatory cytokines and chemokines, such as TNFα, macrophage inflammatory protein-2 (MIP-2), Cyclooxygenase-2 (COX-2) or macrophage inflammatory protein (MIP). These results implicate PKCα in the epidermal inflammatory response (Wang and Smart (1999) J Cell Sci 112:3497-3506). Treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA, a PKCα activator) apparently caused epidermal hyperplasia, intra-epidermal inflammation, and massive apoptosis (Cataisson et al. (2003) J Immunol 171:2703-2713; Jansen et al. (2001) Int J Cancer 93:635-643). In addition, recent in vivo studies in PKC isoenzyme-selective knockout and transgenic mice appear to have highlighted distinct functions of individual PKCs in the immune system. These genetic analyses, along with biochemical studies appear to indicate that PKC-regulated signaling pathways play a significant role in many aspects of the immune responses. For example, members of the PKC family appear crucial in T cell signaling pathways. Particularly, PKCα, isotype appears to determine the nature of lymphocyte-specific in vivo effector. PKCα is also discussed as being involved in macrophages activation and was apparently shown to be involved in mast cell signaling (Cataisson et al. (2005) J Immunol 174:1686-1692).
One example of an inflammatory disease is psoriasis. There are two main hypotheses about the basic pathology leading to psoriasis development. The first considers psoriasis as primarily a disorder of excessive growth and reproduction of skin cells. The second hypothesis considers psoriasis as an immune-mediated disorder in which the excessive reproduction of skin cells is secondary to factors produced by the immune system. Accordingly, most drugs for psoriasis target one component of the disease, either the hyper-proliferative state of skin cells, or the skin inflammatory response as presented in psoriasis plaques.
Recent data support the notion that both pathways underlie the pathology of the diseases through a cross talk between skin cells and immunological milieu (encompassing environment, surroundings, location and/or setting). Classic genome wide linkage analysis has identified nine locations (loci) on different chromosomes associated with tendency to develop psoriasis named psoriasis susceptibility 1 through 9 (PSORS1 through PSORS9) loci. In these locations several genes were characterized and found to encode for proteins expressed in epidermal cells such as corneodesmosin, expressed in the granular and cornified layers of the epidermis and upregulated in psoriasis. On the other hand, other psoriasis linked genes encode for proteins involved in modulation of the immune system where characterized such as IL-12B on chromosome 5q (Frank et al. (2009) N Engl J Med 361:496-509).
WO 2005/007072 of some of the inventors of the present application discloses pharmaceutical compositions for topical administration, for inducing or accelerating a healing process of a damaged skin or skin wound, comprising insulin and additional agent, such as alpha PKC inhibitor, acting in synergy with the insulin.
WO 2009/016629 of some of the inventors of the present application discloses compositions comprising a delta-PKC activator, an alpha PKC inhibitor, and a carrier that is free of calcium (Ca2+) and magnesium (Mg2+) cations for decreasing inflammation at the site of a skin wound.
Current therapies for combating inflammatory diseases generally fail to provide a multi-component approach targeting multiple components of pathogenesis. For example, many treatments for autoimmune diseases involve targeting a single component of a disease, either by blocking cellular proliferation, or by suppressing the immune response in order to block inflammation. Consequently, there is a strong need to provide effective therapeutics which target multiple components of inflammatory disease pathogenesis by targeting and modulating PKC isoform activity. Specifically targeted therapeutics that are capable of selective inhibition or activation of specific PKC isoforms are necessary and would provide for a therapeutic approach that targets multiple components of inflammatory disease pathogenesis, while retaining a low level of side effects, for example, when topically administered. Thus, development of therapeutics that reduce secretion of proinflammatory cytokines and/or regulate immunomodulators via PKC isoform modulation would be beneficial in alleviating topical and systemic inflammation generally and specifically, inflammation of the skin and other epithelial tissues.